DNA damage mediates widespread changes in transcription through activation or repression of transcription factors (TFs). However, the consequences of regulating specific TFs for the outcome of the DNA repair process remain incompletely understood. Here, we combined transcriptomics and TF binding prediction with functional genomics to identify TFs essential for adequate DNA repair in HepG2 liver cells after a non-cytotoxic dose of carcinogens benzo(a)pyrene (BaP) (2μM) and aflatoxin B1 (AFB1) (5μM). BaP and AFB1 induced a largely common transcriptional response, mediated by similar TFs. A lentiviral shRNA screen knocking down the top31 identified TFs, was performed to determine their effect on DNA repair by assessing phosphorylation of H2AX (γ-H2AX). In addition to the top candidate p53, we identified several other interesting TFs that modulated γ-H2AX after BaP and AFB1 treatment. Validation studies confirmed the role of p53 in reducing γ-H2AX formation and DNA breaks measured by COMET assay after BaP and AFB1 exposure. Expression of the cell cycle inhibitor p21 was profoundly impaired upon p53 knock-down. In addition, the expression of 2 genes involved in nucleotide exchange repair, DDB2 and XPC was significantly reduced in p53 knock-down cells. Although p63 knock-down affected DNA damage upon BaP treatment this was not associated with altered expression of DDB2 or XPC. Finally, knock-down of ARNT reduced γ-H2AX in response to BaP, which was associated with reduced CYP1A1 expression. Importantly, our results suggest a new role for ARNT and its dimerization partner AHR in the occurrence of H2AX phosphorylation after AFB1 treatment. These data show that modulation of TF activity impacts on the repair of BaP- and AFB1-induced DNA damage. Our study also demonstrates the potential of combining functional genomics with genome-wide expression analysis to identify yet unknown causal relationships, thereby aiding in the interpretation of complex biological systems.

A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

Valproic acid (VPA) is a frequently prescribed anti-epileptic drug which is known to cause liver toxicity and steatosis through mitochondrial dysfunction. Nevertheless the mechanisms underlying these adverse effects are incompletely understood. In this study, we determined the effect of relatively short (3 h) or prolonged (72 h) exposure to VPA on mitochondrial function in primary human hepatocytes (PHHs). While 3 h VPA exposure did not affect oxygen consumption rates (OCRs) in PHHs, prolonged exposure (24–72 h) significantly reduced basal and maximal OCRs. Given that in particular prolonged VPA exposure is required to cause mitochondrial dysfunction, we investigated gene expression data after VPA exposure for 24, 48, 72 h and 72 h VPA followed by a 72 h washout period. We were able to reduce the comprehensive gene expression changes into a more comprehensible set of 18 TFs that were predicted to be persistently activated after 72 h of VPA exposure. Lentiviral knock-down of one of the candidate TFs, C/EBPα, partly rescued VPA-induced mitochondrial dysfunction. Furthermore, RNA-Seq analysis of shC/EBPα and shGFP control PHHs identified 24 genuine C/EBPα target genes that are regulated in response to prolonged VPA exposure in PHHs. Altogether this provides new insights on the involvement of C/EBPα in driving VPA-induced mitochondrial dysfunction in human liver cells. This hub gene, with its downstream regulators involved in this deregulation, thus represent potential new biomarkers for VPA-induced mitochondrial dysfunction.

The discovery of induced pluripotent stem cell (iPSC) technology has the potential to accelerate scientific research for Alzheimer's disease (AD). iPSCs are therefore increasingly considered for AD modeling and drug development. Nevertheless, most of the work conducted so far has mainly focused on iPSC models from patients with familial AD (fAD), while actually sporadic AD (sAD) is more prevalent and represents over 90% of the AD cases in the population. The development of more sAD models is therefore key for studying this multifactorial disorder. In fact, probing the unique genomes of sAD patients and their interaction with AD-associated environmental factors could contribute to a better understanding of this disease. However, initial iPSC-based models for sAD have shown a high degree of variability and inconsistencies in terms of AD hallmarks. In this review, we provide an overview of the studies that have been conducted for sAD so far. In addition, we critically assess important sources of variability related to the model in addition to those that might be explained by the heterogeneous nature of sAD. These considerations might aid in developing more consistent iPSC models of sAD, which could help in developing a better understanding of the molecular mechanisms underlying the disease.

Recent advances in cancer therapy have substantially increased survival rates among patients, yet the prolonged effect of current treatment regimens with anthracyclines (ACs) often include severe long-term complications, notably in the form of anthracycline-induced cardiotoxicity (AIC). Despite known associations between AC treatment and AIC, a comprehensive understanding of the underlying molecular pathways remains elusive. This gap is highlighted by the scarcity of reliable therapeutic interventions, with dexrazoxane being the sole FDA-approved drug to mitigate AIC risks. This study aims at elucidating the transcriptional response of human cardiomyocytes (hCMs) to AC exposure by analyzing a previously generated RNA-sequencing dataset of cardiac spheroids subjected to clinically relevant doses of ACs. The analysis revealed a robust transcriptional response identified across various time points. We aimed at identifying important transcription factors (TFs) mediating AIC by employing predictive algorithms to highlight key TFs for further experimental validation. Using shRNA constructs, we further assessed the impact of these TFs on hCM response to doxorubicin (DOX) and revealed that these TFs had a notable impact on hCM survival upon DOX exposure. TFs FOXO3, GATA2, AHR and NFE2L2 were further investigated for their role in AIC including cell viability, DOX uptake, DNA damage repair and induction of apoptosis through Cleaved-Caspase 3. Our study demonstrated that eliminating FOXO3 and GATA2 made hCMs more vulnerable to DOX and the lack of GATA2, NFE2L2 and AHR led to significantly higher intracellular levels of DOX. Additionally, FOXO3 played a role in the repair of hCM DNA damage as we observed markedly enhanced levels of CDKN1A. We also noted significant increases in DNA damage through COMET-assays when FOXO3, GATA2, NFE2L2 and AHR were absent. Furthermore, we investigated the clinical relevance by comparing our results with those from a study based on hiPSC-CMs derived from patients with doxorubicin-induced cardiotoxicity, identifying overlapping TFs and their regulatory roles in critical cellular processes like the cell cycle and DNA repair. This approach not only advances the understanding of the molecular mechanisms behind AIC but also opens possible windows for new therapeutic approaches to mitigate the negative side-effects from patient AC treatment.